Breast cancer remains the most common cancer among American women, representing an estimated 30% of all new cancer cases in women. The current staging modalities for breast cancer are invasive, expensive, and lack sensitivity, limiting the ability of clinicians to tailor therapies to individual patients. The identification of genes overexpressed in breast cancer combined with recent advances in molecular biology provide the opportunity to establish more sensitive, specific, and potentially cost-effective ways of staging breast cancer. Two new technologies have recently been developed that significantly enhance the ability to detect breast cancer-associated gene overexpression in the peripheral blood: a novel porous barrier density gradient centrifugation technique, and real-time RT-PCR. The hypothesis of the proposed research is that realtime RT-PCR detection of breast cancer cells in the peripheral blood of breast cancer patients is associated with clinical outcome. In Specific Aim 1, Objective 1 we will establish criteria for defining positive molecular test results. To establish rigorous threshold values for marker positivity, we will obtain peripheral blood samples from a cohort of healthy volunteer subjects with no evidence of malignancy. We will determine the background expression of candidate molecular markers in these specimens. We will also determine if the background expression of candidate markers is independent of baseline patient characteristics including age (>50 versus =50), race (White versus Not White) and the presence of benign breast disease (Present versus Absent). In Objective 2, we will design and validate internal positive control gene fragment mimetics to be used as an additional control for the molecular assays. In Specific Aim 2, we will define the clinical relevance of molecular detection of circulating breast cancer cells in a double-blinded prospective cohort study involving 92 subjects at Washington University in Saint Louis and the Medical University of South Carolina. The primary hypothesis is that Stage IV breast cancer patients initiating a new regimen of systemic therapy with molecular evidence of breast cancer in the peripheral blood will experience a decreased 1-year progression-free and overall survival compared to Stage IV breast cancer patients with no molecular evidence of breast cancer in the peripheral blood. Secondary objectives will address the significance of molecular detection of breast cancer cells after the initiation of therapy. Advances in molecular genetics have brought a revolution to medicine;the successful development of a molecular assay for the detection of breast cancer cells in the peripheral blood of breast cancer patients is likely to do the same for those unfortunate women afflicted with this disease.